High Strength Buffer Research Articles

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates.

Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, and Limulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfused in situ into rat pancreatic vasculature previously oxidized with 1 mM sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.

The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes.

We have examined the relationship of acetylcholine receptors (AChR) to the Mr 43,000 receptor-associated protein (43K) in the AChR clusters of cultured rat myotubes. Indirect immunofluorescence revealed that the 43K protein was concentrated at the AChR domains of the receptor clusters in intact rat myotubes, in myotube fragments, and in clusters that had been purified approximately 100-fold by extraction with saponin. The association of the 43K protein with clustered AChR was not affected by buffers of high or low ionic strength, by alkaline pHs up to 10, or by chymotrypsin at 10 micrograms/ml. However, the 43K protein was removed from clusters with lithium diiodosalicylate or at alkaline pH (greater than 10). Upon extraction of 43K, several changes were observed in the AChR population. Receptors redistributed in the plane of the muscle membrane in alkali-extracted samples. The number of binding sites accessible to an anti-AChR monoclonal antibody directed against cytoplasmic epitopes (88B) doubled. Receptors became more susceptible to digestion by chymotrypsin, which destroyed the binding sites for the 88B antibody only after 43K was extracted. These results suggest that in isolated AChR clusters the 43K protein covers part of the cytoplasmic domain of AChR and may contribute to the unique distribution of this membrane protein.

Time-course and stoichiometry of light-induced proton release and uptake during the photocycle of bacteriorhodopsin

The kinetics and stoichiometry of light-induced proton release in purple membrane suspensions have been investigated using the pH-indicator dye pyranine and single-turnover flash spectroscopy at a time resolution of 0.1 μs. The number of protons detected by pyranine is inversely proportional to the buffering power of the medium and 1.1 ± 0.2 protons are released per photocycling bacteriorhodopsin molecule (pH 7.0, 20°C). At high ionic strength (250 mM KCl) and higher phosphate buffer concentrations (1 mM) the proton release lags slightly behind the formation of the intermediate M-412 and proton reuptake by the purple membrane parallels the slow decay process of M-412 or the decay of O-640.

Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts.

Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.

Propylbenzilylcholine mustard is selective for rat heart muscarinic receptors having a low affinity for agonists.

Propylbenzilylcholine mustard (PrBCM), an irreversible muscarinic antagonist, inactivated receptors with a low affinity for agonists faster than those with a high affinity in rat heart membranes. This result was obtained using either: (a) a low ionic strength buffer (allowing heterogeneity among antagonist binding sites, (b) the same buffer enriched with GTP, or (c) a high ionic strength buffer (where antagonists showed similar binding characteristics to all receptors). These data suggest either that PrBCM is a 'selective' antagonist which detects conformational differences between low and high affinity receptors, or that the agonist affinity of cardiac muscarinic receptors is determined, in part, by the relative concentrations of receptor and GTP binding protein.

Formation of the thiol adducts of 4′-(9-acridinylamino)methanesulfon- m-anisidide and their binding to deoxyribonucleic acid

We investigated the interactions of 4′-(9-acridinylamino)methanesulfon- m-anisidide (mAMSA) with thiol-containing compounds and the potential binding of the thiolytic adducts to DNA. All thiols tested (glutathione, cysteine, coenzyme A, 2-mercaptoethanol and lactate dehydrogenase) formed adducts with mAMSA as evidenced by changes in the absorption spectrum of mAMSA and induction of fluorescence. Spectral changes induced by the thiols were different, suggesting that each thiol induced specific changes in the electronic structure of the acridine nucleus. Treatment of glutathione with p-chloromercuribenzoate eliminated the absorption spectral changes and induction of fluorescence, indicating that the reduced-thiol group is involved. In high ionic strength buffer, addition of calf thymus DNA induced fluorescence-quenching of both the mAMSA-glutathione and mAMSA-cysteine adducts without spectral shift. Viscometric studies showed that mAMSA and mAMSA-glutathione intercalated into DNA and produced similar increases in the length of linear DNA.

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix

Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pI of 8.7-8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma.

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.

Analysis of monomeric-dimeric states of the estrogen receptor with monoclonal antiestrophilins

We studied the antibody-combining properties of 3 forms of the estrogen receptor found in buffers of high ionic strength. Shifts to a faster sedimenting peak on sucrose gradients or a faster eluting peak on a gel filtration column with antibody addition allowed us to determine whether a given form contained one, two or more antibody-binding sites. The monomeric cytosolic estrogen receptor, ER C, contained one antibody binding site for each of 2 monoclonal antiestrophilins (H222 and H165, provided by Abbott Laboratories). Both the heat-transformed cytosolic estrogen receptor, ER ∗ C, and a major fraction of the estrogen receptor extracted from nuclei, ER N, contained two sites for H165, but only one for H222. A minor fraction of ER N had only one site for each antibody. The kinetics of transformation of ER C. to a species with two H165 binding sites were appropriate to a dimerization of ER C. Addition of H222, but not H165, before the onset of the heat-induced transformation blocked the formation of ER C to ER C ∗. These data suggest that ER C ∗ and a major form of ER N are comprised of two immunologically similar subunits identical to ER C. Also, the antigenic determinant for H222, but not H165, appears to be located close to the dimerization domain. The minor form of ER N appears to contain an altered or dissimilar subunit.

Regulation of the NAD Malic Enzyme from Crassula

Using size exclusion chromatography, the nicotinamide adenine dinucleotide malic enzyme purified to near homogeneity from leaves of Crassula argentea was found to exist in at least three aggregational states (dimer, tetramer, and octamer). These forms differ in their apparent kinetic characteristics in initial rate assays, but all display similar characteristics at the steady state. The presence of 50 millimolar malate during chromatography causes a shift in favor of the smaller forms with the tetramer predominating. The native enzyme, when diluted 1/1000 and incubated 18 hours in buffer of high ionic strength, changes its steady state kinetic parameters to ones which indicate a low activity and low affinity for malate. When 50 millimolar malate or 50 micromolar coenzyme A are present the loss of activity and increase in K(m) is reduced. When both malate and coenzyme A are present the effects in minimizing the change in kinetic characteristics are additive.

Purification and characterization of GTP cyclohydrolase I from Drosophila melanogaster.

The enzyme GTP cyclohydrolase I, which catalyzes the first step in the pteridine biosynthetic pathway, has been purified by at least 4400-fold from Drosophila melanogaster. The active complex has an apparent molecular mass of 575,000 daltons, as estimated from gel filtration. This high molecular mass complex appears to be composed of a number of 39,000-dalton subunits. A polyspecific antiserum generated against the active complex has been used to identify this polypeptide as being severely affected by mutations in Punch, the structural gene for GTP cyclohydrolase. Enzyme activity is inhibited by divalent cations and high ionic strength buffers. No cofactors have been demonstrated to be required for enzyme activity. The enzyme displays positive cooperativity in phosphate buffer, a Hill number of 2.1, but only slight cooperativity in Tris buffer, a Hill number of 1.2.

37] Affinity chromatography of heme-binding proteins: synthesis of hemin-agarose

This chapter describes the affinity chromatography of heme-binding proteins and the synthesis of hemin–agarose. Some features of the method for preparing hemin–agarose may be described as follows: (1) to avoid the solubility problem, the coupling reaction is performed in a pure nonaqueous solvent (100% DMF) in which hemin is highly soluble, (2) the immobilization is accomplished through the propionic acid group of hemin, (3) to activate the carboxyl group, 1, l ' -carbonyldiimidazole (CDI) is employed instead of more commonly used agents such as carbodiimides. This agent (CDI) is more convenient to use because there is no need to adjust the pH during the reaction, (4) a cross-linked agarose (Sepharose CL-6B) which is quite resistant to organic solvents and mechanical agitation is used as the matrix, (5) the time required to remove the unreacted hemin which is tightly adsorbed on the hemin–agarose is significantly reduced by introducing a wash with 25% pyridine, and (6) compared to other methods, a significantly higher coupling efficiency (1–2.5 μ mol hemin/ml agarose) is obtained. Some properties of the hemin–agarose resin prepared by the present synthetic procedure but using an uncross-linked agarose matrix have been described. Intactness of the ligand during the coupling reaction is demonstrated by the identical pyridine hemochrome absorption spectra of hemin and the hemin derivative released from the resin by alkali treatment. For affinity chromatography on the hemin–agarose, a high-ionic strength buffer (0.5 M NaCl and 10 mM sodium phosphate, pH 7.5) has been routinely used in the protein adsorption step to minimize nonspecific ionic interaction between protein and the affinity resin. The chapter also discusses the affinity chromatography of serum heme-binding proteins on hemin–agarose.

Tandem purification of mouse IgM monoclonal atibodies produced in vitro using anion-exchange and gel fast protein liquid chromatography

A tande chromatographic procedure was used to isolate rapidly mouse IgM monclonal antibodies produced by cultivation of hybridomas in vitro. Hybridoma culture supernatants containing mouse IgM monoclonal antibodies were first chromatographed on an anion-exchange Mono Q column connected to a fast protein liquid chromatography system. This anion-exchange step offers the advantage of obtaining IgM antibodies in a concentrated form The IgM-rich fractions from the Mono Q column were then injected on a gel filtration Superose 6 column equilibrated with a low-ionic strength buffer and eluted with a high-ionic strength buffer. Assessment of the purity of isolated IgM monoclonal antibodies was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis together with a Coomassie Brillant Blue r 250 staining technique. Assessment of the immunoreactivity of isolated IgM monoclonal antibodies was performed by an enzyme linked immunosorbent assay using a solid phase adsorbed antigen against which IgM monoclonal antibodies were directed. The chromatographic procedures described allows the rapid isolation of mouse IgM monoclonal antibodies produced in vitro at a high degree of purity and in an immunoreactive state.

Solubilization and assay of a colony‐stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).

A network of 2–4 nm filaments found in sea urchin smooth muscle: Protein constituents and in situ localization

In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights ( M r ) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2–4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2–4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed.

Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease

In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to [ 3H]4-OHTAM. The [ 3H]4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea. The following parameters were determined: sedimentation coefficients (S) on a sucrose gradient. Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Following mild nuclease digestion, the solubilized [ 3H]-OHTAM bound receptor sedimented as an abundant 6–7 S form and a less abundant ~ 12 S species. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000. About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer. Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000. Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000. These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexed with DNA.

Androgen directs apparent cytoplasmic and nuclear distribution of rat cardiovascular androgen receptors.

We used either the synthetic androgen R1881 (methyltrienolone) or 5 alpha-dihydrotestosterone (5 alpha-DHT) to characterize androgen receptors in rat aortic and myocardial cytoplasmic and nuclear preparations. Relative steroid specificity studies established that only androgens were effective inhibitors of R1881 (cytoplasmic) or 5 alpha-DHT (nuclear) binding to aortic and myocardial androgen receptors, whereas estrogens, progestins, and cortisol were ineffective inhibitors. Low ionic strength sucrose density gradient centrifugation analyses showed that androgen receptors in aortic and myocardial cytoplasmic preparations migrated as macromolecules with sedimentation coefficients of 8 to 9S, whereas androgen receptors in aortic and myocardial nuclear preparations migrated as macromolecules with sedimentation coefficients of 4 to 5S (high ionic strength buffer). Saturation analyses established that aortic and myocardial cytoplasmic preparations from intact, untreated young mature female rats contained 45.8 +/- 9.7 (mean +/- SD) and 63.3 +/- 20.7 fmol androgen receptor/mg DNA, respectively. The respective R1881 dissociation constants were 0.60 and 0.32 nM. Androgen receptors could not be demonstrated in nuclear preparations from the cardiovasculature of intact females. Testosterone injection of intact young mature female rats caused apparent depletion of androgen receptors in aortic and myocardial cytoplasmic preparations and resulted in concomitant appearance of 57.1 +/- 22.2 and 52.3 +/- 21.5 fmol receptor/mg DNA in the corresponding nuclear preparations. The respective 5 alpha-DHT dissociation constants were 4.46 and 1.63 nM. The ability of testosterone to affect apparent intracellular distribution of cardiovascular androgen receptors suggests that the receptors are physiologically functional and indicates that androgen may directly regulate cardiovascular cell function.

Effect of swimming training on cardiac function and myosin ATPase activity in SHR.

Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) were subjected to swimming training 6 times/wk, commencing at 4 wk of age, to determine whether this type of endurance exercise might alter contractile proteins and cardiac function in young adult SHR. The total duration of exercise was 190 h. Myofibrillar adenosinetriphosphatase (ATPase) activity was assayed at various free [Ca2+] ranging from 10(-7) to 10(-5) M. Ca2+-stimulated ATPase activity of actomyosin and purified myosin was determined at various Ca2+ concentrations both in the low and high ionic strength buffers. Actin-activated myosin ATPase activity of purified myosin was assayed at several concentrations of actin purified from rabbit skeletal muscle. Under all these conditions the contractile protein ATPase activity was comparable between trained and untrained WKY and SHR. Analysis of myosin isoenzymes on pyrophosphate gels showed a single band corresponding to V1 isoenzyme, and there were no differences between swimming-trained and nontrained WKY and SHR. Ventricular performance was assessed by measuring cardiac output and stroke volume after rapid intravenous volume overloading. Both cardiac index and stroke index were comparable in nontrained WKY and SHR but were significantly increased in the trained groups compared with their respective nontrained controls. These results suggest that myosin ATPase activity and distribution of myosin isoenzymes are not altered in the moderately hypertrophied left ventricle whether the hypertrophy is due to genetic hypertension (SHR) or to exercise training (trained WKY). Moreover, the data indicate that SHR, despite the persistence of a pressure overload, undergo similar increases in left ventricular mass and peak cardiac index after training, as do normotensive WKY.

Binding of complement component C1q by rat adipocyte membranes

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.

Properties and partial purification of infective material from plants containing groundnut rosette virus*

SUMMARYGroundnut plants with chlorotic rosette disease contain a manually transmissible virus, groundnut rosette (GRV), which is also transmitted in the persistent (circulative) manner by aphids (Aphis craccivora), but only from plants that are co‐infected with a manually non‐transmissible luteovirus, groundnut rosette assistor virus (GRAV). Strains of GRV from plants with chlorotic or green forms of rosette are called GRV(C) and GRV(G) respectively. An isolate of GRV(C) from Nigeria remained infective in Nicotiana clevelandii leaf extracts for 1 day at room temperature and for 15 days at 4d̀C, but lost infectivity after 1 day at ‐20d̀C or after dilution to 10‐‐4. Its infectivity and longevity in vitro were not altered by addition of 1 mg/litre bentonite to the extraction buffer. Infectivity in leaf extracts was abolished by treatment with 50% (v/v) ether, 10% (v/v) chloroform or 8% (v/v) n‐butanol, but not by treatment for 30 min with RNase A at up to 100 ng/ml. In attempts to purify GRV(C), nearly all the infectivity from N. clevelandii extracts was found in the pellets from centrifugation at 65 000 g for 1. 5 h; infectivity also occurred in a cell membrane fraction that collected at the top of a 30% sucrose ‘cushion’ containing 4% polyethylene glycol and 0.2 M NaCI. However, no virus‐like particles were found in either type of preparation by electron microscopy.Nucleic acid preparations made directly from GRV(C)‐infected N. clevelandii leaves were very infective; this infectivity was totally inactivated by treatment for 30 min with RNase A at 10 ng/ml in buffers of both low and high ionic strength and was therefore attributed to ssRNA. When nucleic acid preparations were electrophoresed in gels no virus‐specific bands were visible but the position of the infectivity indicated that the infective ssRNA has an apparent mol. wt of c. 1.55 × 106. A similar mol. wt was indicated by the rate of sedimentation of the infective ssRNA in sucrose gradients. Preparations of dsRNA made from GRV(C)‐infected N. clevelandii leaves contained a species of mol. wt c. 3.0 × 106; in addition some dsRNA preparations contained an abundant component of mol. wt c. 0.6 × 106 together with several other components of intermediate mol. wt. Similar patterns of bands were observed in dsRNA preparations made from Nigerian‐grown groundnut material infected with GRV(C) alone, or with GRV(C) + GRAV, or with GRV(G) + GRAV.The properties of GRV closely resemble those of two other viruses that depend on luteoviruses for transmission by aphids, carrot mottle virus and lettuce speckles mottle virus.